In order to understand brain functions, it is first necessary to observe its physiological functions. Information processing of nerve cells that support the higher functions of the brain is conducted by a change in electrical potential in a cell membrane and, therefore, since development of the patch clamp technique, the measurement of the electrical potential in a cell membrane has been mainly carried out using the electrophysiological technique.
Brain tissues are formed by a network in which a large number of nerve cells and glial cells are combined in a complex way and each cell also has a very complicated structure, and observation by an electrophysiological technique was therefore restricted. For example, since many of spines that are considered to be crucial for information processing and fine dendrites that support the spines have very fine structures with a diameter of less than 1 micron, the size of a glass electrode becomes a problem for the spines and dendrites and they are not suitable for observation by the above described electrophysiological technique.
Therefore, the measurement and visualization of electrical potential in a membrane using an optical technique have been tried in place of an electrophysiological technique (Patent Documents 1 to 5). Many imaging procedures on the basis of observation of fluorescence from electrical potential sensitive dyes and green fluorescent proteins (GFP) have been developed so far; however, have two large problems regarding time resolution ability and quantitativity. The utilization of second harmonic generation (SHG) imaging has been proposed as a technique capable of overcoming these problems, (Patent Documents 4 and 5).
SHG is a two-photon phenomenon in which two photons interact with a non-centrosymmetric substance and then transferred to the same direction as the incident angle as one photon having doubled energy. Observation utilizing SHG can use high-tissue-permeability near-infrared light from a two-photon microscope and thus it is suitable for observation of a deep part of a tissue. In observation utilizing a fluorescent dye, the dye absorbs one or two photons to be excited and, after relaxation, returns to the ground state, a quantitative measurement is fundamentally impossible; on the contrary, quantitative visualizations of membrane electrical potential that do not depend on cell form can be possible using this SHG imaging. In addition, the capability of preventing damage to a living tissue, which is caused by excitation of a dye, is also one of advantages.
When FM4-64, which is a known florescent dye, is introduced into a nerve cell from the cell exterior and irradiated with a femtosecond laser, a very strong SHG signal is detected by a detector in a light transmitted path in addition to a fluorescent (TPF) signal (Patent Document 4). Employment of this method enabled observation of fine structures such as distal dendrites and spines. Furthermore, it was also confirmed that when membrane electrical potential in a cell is changed by a membrane electrical potential fixing method, a SHG signal is accordingly reliably changed.
Patent Document 1: Japanese Unexamined Patent Application, Publication No. H08-122326
Patent Document 2: Japanese Unexamined Patent Application, Publication No. H09-005243
Patent Document 3: Japanese Unexamined Patent Application (Translation of PCT Application), Publication No. H11-508355
Patent Document 4: Japanese Unexamined Patent Application, Publication No. 2004-212132
Patent Document 5: Japanese Unexamined Patent Application, Publication No. 2012-14066